Lettuce variety 45-ic3284 rz

ABSTRACT

The present invention relates to a Lactuca sativa seed designated 45-IC3284 RZ. The present invention also relates to a Lactuca sativa plant produced by growing the 45-IC3284 RZ seed. The invention further relates to methods for producing the lettuce cultivar, represented by lettuce variety 45-IC3284 RZ.

INCORPORATION BY REFERENCE

This application claims priority to U.S. provisional patent applicationSer. No. 63/299,592 filed 14 Jan. 2022.

The foregoing applications, and all documents cited therein or duringtheir prosecution (“appln cited documents”) and all documents cited orreferenced in the application cited documents, and all documents citedor referenced herein (“herein cited documents”), and all documents citedor referenced in herein cited documents, together with anymanufacturer's instructions, descriptions, product specifications, andproduct sheets for any products mentioned herein or in any documentincorporated by reference herein, are hereby incorporated herein byreference, and may be employed in the practice of the invention. Morespecifically, all referenced documents are incorporated by reference tothe same extent as if each individual document was specifically andindividually indicated to be incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to a new lettuce (Lactuca sativa) varietydesignated 45-IC3284 RZ. Lettuce variety 45-IC3284 RZ exhibits acombination of traits including resistance to downy mildew (Bremialactucae) races B1:16EU to B1:37EU and B1:1US to B1:9US, resistance tocurrant-lettuce aphid (Nasonovia ribisnigri) biotype Nr:0, resistance toLettuce mosaic virus (LMV), intermediate resistance to Fusariumoxysporum f sp. lactuace race 4 (Fol:4), resistance to lettuce rootaphids (pb) (Pemphigus bursarius), as well as having mature leaves withmedium blistering.

BACKGROUND OF THE INVENTION

All cultivated forms of lettuce belong to the highly polymorphicspecies, Lactuca sativa, which is grown for its edible head and leaves.As a crop, lettuces are grown commercially wherever environmentalconditions permit the production of an economically viable yield.

Lactuca sativa is in the Cichoreae tribe of the Asteraceae (Compositae)family. Lettuce is related to chicory, sunflower, aster, scorzonera,dandelion, artichoke and chrysanthemum. Sativa is one of about 300species in the genus Lactuca.

Lettuce cultivars are susceptible to a number of pests and diseases suchas downy mildew (Bremia lactucae). Every year this disease leads tomillions of dollars of lost lettuce crop throughout the world. Downymildew (Bremia lactucae) is highly destructive on lettuce grown atrelatively low temperature and high humidity. Downy mildew is caused bya fungus, Bremia lactucae, which can be one of the following strains:B1:16EU, B1:17EU, B1:18EU, B1:20EU, B1:21EU, B1:22EU, B1:23EU, B1:24EU,B1:25EU, B1:26EU, B1:27EU, B1:29EU, B1:30EU, B1:31EU, B1:32EU, B1:33EU,B1:34EU, B1:35EU, B1:36EU, B1:37EU (Van Ettekoven, K. et al.,“Identification and denomination of ‘new’ races of Bremia lactucae,” In:Lebeda, A. and Kristkova, E (eds.), Eucarpia Leafy Vegetables, 1999,Palacky University, Olomouc, Czech Republic, pp. 171-175; Van der Arendet al. “Identification and denomination of “new” races of Bremialactucae in Europe by IBEB until 2002″ In: Van Hintum, Th et al. (eds.),Eucarpia Leafy Vegetables Conference 2003, Centre for Genetic Resources,Wageningen, The Netherlands, p. 151; Plantum NL (Dutch association forbreeding, tissue culture, production and trade of seeds and youngplants), Van der Arend et al. “Identification and denomination of “new”races of Bremia lactucae in Europe by IBEB until 2002.” In: Van Hintum,Th et al. (eds.), Eucarpia Leafy Vegetables Conference 2003, Centre forGenetic Resources, Wageningen, The Netherlands, p. 151; Plantum NL; IBEBpress release “New race of Bremia lactucae B1:27 identified andnominated”, May 2010; Plantum NL, IBEB press release, “New races ofBremia lactucae, B1:29, B1:30 and B1:31 identified and nominated”,August 2013; Plantum NL, IBEB press release, “A new race of Bremialactucae, B1:32 identified and nominated in Europe”, May 2015); PlantumNL, IBEB-EU press release, “A new race of Bremia lactucae, B1:33EUidentified and denominated in Europe”, May 2017; Plantum NL, IBEB-EUpress release, “Two new races of Bremia lactucae, B1:34EU and B1:35EUidentified and nominated in Europe”, July 2018; Plantum NL, IBEB-EUpress release, “A new race of Bremia lactucae, B1:36EU identified anddenominated in Europe”, July 2019; Plantum NL, IBEB EU press release, “Anew reace of Bremia lactuceae, B1:37EU identified and denominated inEurope, June 2021), B1:1US, B1:2US, B1:3US, B1:4US, B1:5US, B1:6US,B1:7US, B1:8US, B1:9US (Schettini, T. M., Legg, E. J., Michelmore, R.W., 1991. Insensitivity to metalaxyl in California populations of Bremialactucae and resistance of California lettuce cultivars to downy mildew.Phytopathology 81(1). p. 64-70; Michelmore R. & Ochoa. O. “BreedingCrisphead Lettuce, In: California Lettuce Research Board, Annual Report2005-2006, 2006, Salinas, Calif., pp. 55-68;http://bremia.ucdavis.edu/dataB1_9 US.pdf).

Downy mildew causes pale, angular, yellow areas bounded by veins on theupper leaf surfaces. Sporulation occurs on the opposite surface of theleaves. The lesions eventually turn brown, and they may enlarge andcoalesce. These symptoms typically occur first on the lower leaves ofthe lettuce, but under ideal conditions may move into the upper leavesof the head. When the fungus progresses to this degree, the head cannotbe harvested. Less severe damage requires the removal of more leavesthan usual, especially when the lettuce reaches its final destination.

Of the various species of aphids that feed on lettuce, thecurrant-lettuce aphid (Nasonovia ribisnigri) is the most destructivespecies because it feeds both on the leaves of the lettuce as well asthe heart of the lettuce, making it difficult to control withconventional insecticides. The lettuce aphid feeds by sucking sap fromthe lettuce leaves. Although direct damage to the lettuce may belimited, its infestation has serious consequences because the presenceof aphids makes lettuce unacceptable to consumers.

Lettuce mosaic virus (LMV) mainly infects lettuce seeds, which is theprimary way that the virus is introduced to lettuce in the fields, butalso can infect numerous crops and weeds, thereby creating reservoirs ofthe virus. LMV also can be vectored by aphids, which spread the viruswithin a lettuce field and introduce it into lettuce fields frominfected weeds and crops outside the field.

Symptoms of LMV vary greatly. Leaves of plants that are infected at ayoung stage are stunted, deformed and (in some varieties) show a mosaicor mottling pattern. Such plants rarely grow to full size; head lettucevarieties infected early fail to form heads. Plants that are infectedlater in the growth cycle show a different set of symptoms. These plantsmay reach full size, but the older outer leaves turn yellow, twisted,and otherwise are deformed. On head lettuce, the wrapper leaves oftenwill curve back away from the head and developing heads may be deformed.In some cases, brown necrotic flecks occur on the wrapper leaves.

Lettuce root aphids (Pemphigus bursarius) form clustered colonies onlettuce roots, covering them with a white powdery wax. Feeding on theroots cause the plants to wilt during the day. Individually affectedrootlets turn brown and die. The developing heads remain soft, may failto develop, and yields thus become reduced. Prolonged periods ofinfestation can cause the collapse and death of the plant.

Fusarium root rot (wilt) of lettuce, caused by the soil borne fungusFusarium oxysporum f. sp. lactucae, is an important disease of lettucein warmer growing areas. It is found in the lettuce winter productionareas of Arizona and California, and causes significant damage in theearly plantings. In recent years, Fusarium root rot has also spread tothe summer production area of the Salinas Valley. The fungus maypenetrate plants through natural apertures or via wounds on the roots.Transmission of Fusarium oxysporum f. sp. lactucae may occur throughinfected seeds, infected transplants, or spread by soil as spores mayremain viable in soil for long periods of time. Affected plants exhibitleaf yellowing and wilting. Crown tissue and upper roots display signsof reddish brown necrosis, followed by decaying. Leaf veins may alsoshow vascular necrosis. Infected plants are stunted and often die,resulting in significant crop losses, and unmarketable product. Fusariumroot rot can be prevented by soil sterilisation, however, this is anexpensive process.

Citation or identification of any document in this application is not anadmission that such document is available as prior art to the presentinvention.

SUMMARY OF THE INVENTION

Given the need expressed by relevant stakeholders for a lettuce varietywhich shows a combination of traits including resistance to downy mildew(Bremia lactucae) races B1:16EU to B1:37EU and B1:1US to B1:9US,resistance to currant-lettuce aphid (Nasonovia ribisnigri) biotype Nr:0,resistance to Lettuce mosaic virus (LMV), intermediate resistance toFusarium oxysporum f. sp. lactuace race 4 (Fol:4), resistance to lettuceroot aphids pb (Pemphigus bursarius), as well as having mature leaveswith medium blistering, the present invention addresses this need byproviding a new type of lettuce (Lactuca sativa) variety, designated45-IC3284 RZ.

The present invention provides a new lettuce (Lactuca sativa) varietydesignated 45-IC3284 RZ. This new lettuce variety exhibits a combinationof traits including resistance to downy mildew (Bremia lactucae) racesB1:16EU to B1:37EU and B1:1US to B1:9US, resistance to currant-lettuceaphid (Nasonovia ribisnigri) biotype Nr:0, resistance to Lettuce mosaicvirus (LMV), intermediate resistance to Fusarium oxysporum f sp.lactuace race 4 (Fol:4), resistance to lettuce root aphids pb (Pemphigusbursarius), as well as having mature leaves with medium blistering.

The present invention provides seed of a lettuce (Lactuca sativa)variety designated 45-IC3284 RZ. A sample of seeds of said lettucevariety, have been deposited with the National Collections ofIndustrial, Marine and Food Bacteria (NCIMB) in Bucksburn, Aberdeen AB219YA, Scotland, UK and have been assigned NCIMB Accession No. 44097.

In one embodiment, the invention provides a lettuce plant grown from theseed of lettuce (Lactuca sativa) variety 45-IC3284 RZ.

In another embodiment, the invention provides a lettuce plant designated45-IC3284 RZ, which is a plant grown from seed having been depositedunder NCIMB Accession No. 44097.

In one embodiment, the invention provides for a lettuce plant which maycomprise genetic information for exhibiting all of the physiological andmorphological characteristics of a plant of the invention, wherein thegenetic information is as contained in a plant, a sample of seed of saidvariety having been deposited under NCIMB Accession No. 44097.

In one embodiment, the invention provides for a lettuce plant exhibitingall the physiological and morphological characteristics of a plant ofthe invention, and having the genetic information for so exhibiting thecombination of traits, wherein the genetic information is as containedin a plant, a sample of seed of said variety having been deposited underNCIMB Accession No. 44097.

In an embodiment of the present invention, there also is provided partsof a lettuce plant of the invention, which may include parts of alettuce plant exhibiting all the physiological and morphologicalcharacteristics of a plant of the invention, or parts of a lettuce planthaving any of the mentioned resistance(s) and a combination of traitsincluding one or more or all morphological and physiologicalcharacteristics tabulated herein, including parts of lettuce variety45-IC3284 RZ, wherein the plant parts are involved in sexualreproduction, which include, without limitation, microspores, pollen,ovaries, ovules, embryo sacs or egg cells and/or wherein the plant partsare suitable for vegetative reproduction, which include, withoutlimitation, cuttings, roots, stems, cells or protoplasts and/or whereinthe plant parts are tissue culture of regenerable cells in which thecells or protoplasts of the tissue culture are derived from a tissuesuch as, for example and without limitation, leaves, pollen, embryos,cotyledon, hypocotyls, meristematic cells, roots, root tips, anthers,flowers, seeds or stems. The plants of the invention from which suchparts may come from include those wherein a sample of seed of whichhaving been deposited under NCIMB Accession No. 44097 or lettuce varietyor cultivar designated 45-IC3284 RZ, as well as seed from such a plant,plant parts of such a plant (such as those mentioned herein) and plantsfrom such seed and/or progeny of such a plant, advantageously progenyexhibiting such combination of such traits, each of which, is within thescope of the invention; and such combination of traits.

In a further embodiment there is a plant regenerated from theabove-described plant parts or regenerated from the above-describedtissue culture. Advantageously such a plant may have morphologicaland/or physiological characteristics of lettuce variety 45-IC3284 RZand/or of a plant grown from seed, a sample of seed of which having beendeposited under NCIMB Accession No. 44097—including without limitationsuch plants having all of the physiological and morphologicalcharacteristics of lettuce variety 45-IC3284 RZ and/or of a plant grownfrom seed, a sample of seed of which having been deposited under NCIMBAccession No. 44097.

Accordingly, in still a further embodiment, there is provided a lettuceplant having all of the physiological and morphological characteristicsof lettuce variety 45-IC3284 RZ, a sample of seed of which having beendeposited under NCIMB Accession No. 44097. Such a plant may be grownfrom the seeds, regenerated from the above-described plant parts, orregenerated from the above-described tissue culture. A lettuce planthaving any of the aforementioned resistance(s), and one or moremorphological or physiological characteristics recited or tabulatedherein, and a lettuce plant advantageously having all of theaforementioned resistances and the characteristics recited and tabulatedherein, are preferred. Parts of such plants—such as those plant partsabove-mentioned—are encompassed by the invention.

In a further aspect, the invention provides a method of vegetativelypropagating a plant of lettuce variety 45-IC3284 RZ which may comprise(a) collecting tissue capable of being propagated from a plant oflettuce 45-IC3284 RZ, a sample of seed of said variety having beendeposited under NCIMB Accession No. 44097 and (b) cultivating the tissueto obtain proliferated shoots and rooting the proliferated shoots toobtain rooted plantlets. Optionally the invention further may comprisegrowing plants from the rooted plantlets. Plantlets and plants producedby these methods, are encompassed by the invention.

In one embodiment, there is provided a method for producing a progeny oflettuce cultivar 45-IC3284 RZ which may comprise crossing a plantdesignated 45-IC3284 RZ with itself or with another lettuce plant,harvesting the resultant seed, and growing said seed.

In a further embodiment, a progeny plant is provided which is producedby this method, wherein said progeny exhibits a combination of traitsincluding resistance to downy mildew (Bremia lactucae) races B1:16EU toB1:37EU and B1:1US to B1:9US, resistance to currant-lettuce aphid(Nasonovia ribisnigri) biotype Nr:0, resistance to Lettuce mosaic virus(LMV), intermediate resistance to Fusarium oxysporum f sp. lactuace race4 (Fol:4), resistance to lettuce root aphids pb (Pemphigus bursarius),as well as having mature leaves with medium blistering.

In another embodiment, a progeny plant is provided which is produced bythe above method, wherein said progeny exhibits all the morphologicaland physiological characteristics of the lettuce variety designated45-IC3284 RZ, a sample of seed of said variety having been depositedunder NCIMB Accession No. 44097.

In a further embodiment there is provided a progeny plant produced bysexual or vegetative reproduction, grown from seeds, regenerated fromthe above-described plant parts, or regenerated from the above-describedtissue culture of the lettuce cultivar or a progeny plant thereof, asample of seed of which having been deposited under NCIMB Accession No.44097. The progeny may have any of the aforementioned resistance(s), andone or more morphological or physiological characteristics recited ortabulated herein, and a progeny plant advantageously having all of theaforementioned resistances and the characteristics recited and tabulatedherein, are preferred.

Progeny of the lettuce variety 45-IC3284 RZ may be modified in one ormore other characteristics, in which the modification is a result of,for example and without limitation, mutagenesis or transformation with atransgene.

In still another embodiment, the present invention provides progeny oflettuce cultivar 45-IC3284 RZ produced by sexual or vegetativereproduction, grown from seed, regenerated from the above-describedplant parts, or regenerated from the above-described tissue culture ofthe lettuce cultivar or a progeny plant thereof.

The invention further relates to a method for producing a seed of a45-IC3284 RZ-derived lettuce plant which may comprise (a) crossing aplant of lettuce variety 45-IC3284 RZ, a sample of seed of which havingbeen deposited under NCIMB Accession No. 44097, with a second lettuceplant, and (b) whereby seed of a 45-IC3284 RZ-derived lettuce plantforms. This method may further comprise (c) crossing a plant grown from45-IC3284 RZ-derived lettuce seed with itself or with a second lettuceplant to yield additional 45-IC3284 RZ-derived lettuce seed, (d) growingthe additional 45-IC3284 RZ-derived lettuce seed of step (c) to yieldadditional 45-IC3284 RZ-derived lettuce plants, and (e) repeating thecrossing and growing of steps (c) and (d) for an additional 3-10generations to generate further 45-IC3284 RZ-derived lettuce plants, and(f) whereby seed of a 45-IC3284 RZ-derived lettuce plant forms. A seedproduced by this method and a plant grown from said seed also form partof the invention.

The invention also relates to a method of introducing at least one newtrait into a plant of lettuce variety 45-IC3284 RZ which may comprise:(a) crossing a plant of lettuce variety 45-IC3284 RZ, a sample of seedof which having been deposited under NCIMB Accession No. 44097, with asecond lettuce plant that may comprise at least one new trait to produceprogeny seed, (b) harvesting and planting the progeny seed to produce atleast one progeny plant of a subsequent generation, wherein the progenyplant may comprise the at least one new trait, (c) crossing the progenyplant with a plant of lettuce variety 45-IC3284 RZ to produce backcrossprogeny seed, (d) harvesting and planting the backcross progeny seed toproduce a backcross progeny plant, and (e) repeating steps (c) and (d)for at least three additional generations to produce a lettuce plant ofvariety 45-IC3284 RZ which may comprise at least one new trait and allof the physiological and morphological characteristics of a plant oflettuce variety 45-IC3284 RZ, when grown in the same environmentalconditions. A lettuce plant produced by this method also forms part ofthe invention.

Mutations can be introduced randomly by means of one or more chemicalcompounds, such as ethyl methane sulphonate (EMS), nitrosomethylurea,hydroxylamine, proflavine, N-methly-N-nitrosoguanidine,N-ethyl-N-nitrosourea, N-methyl-N-nitro-nitrosoguanidine, diethylsulphate, ethylene imine, sodium azide, formaline, urethane, phenol andethylene oxide, and/or by physical means, such as UV-irradiation, fastneutron exposure, X-rays, gamma irradiation, and/or by insertion ofgenetic elements, such as transposons, T-DNA, retroviral elements.

Mutations can also be introduced by more specific, targeted introductionof at least one modification by means of homologous recombination,oligonucleotide-based mutation introduction, zinc-finger nucleases(ZFN), transcription activator-like effector nucleases (TALENs) orClustered Regularly Interspaced Short Palindromic Repeat (CRISPR)systems.

The invention further relates to a method of producing a plant oflettuce variety 45-IC3284 RZ which may comprise at least one new trait,the method which may comprise introducing a mutation or transgeneconferring the at least one new trait into a plant of lettuce variety45-IC3284 RZ, wherein a sample of seed of said variety has beendeposited under NCIMB Accession No. 44097. A lettuce plant produced bythis method also forms part of the invention.

In still a further embodiment, the invention provides a method ofproducing a lettuce seed which may comprise crossing a male parentlettuce plant with a female parent lettuce plant and harvesting theresultant lettuce seed, in which the male parent lettuce plant or thefemale parent lettuce plant is a lettuce plant of the invention, e.g. alettuce plant having all of the morphological or physiologicalcharacteristics tabulated herein, including a lettuce plant of lettucecultivar 45-IC3284 RZ, a sample of seed of which having been depositedunder 44097. The resultant lettuce seed produced by this method and thelettuce plant that is produced by growing said lettuce seed also formspart of the invention.

In still a further embodiment, the invention provides a method ofproducing a lettuce cultivar which exhibits all of the physiological andmorphological characteristics of lettuce variety 45-IC3284 RZ, a sampleof seed of said variety having been deposited under NCIMB Accession No.44097.

The invention even further relates to a method of producing lettuceleaves as a food product which may comprise: (a) sowing a seed oflettuce variety 45-IC3284 RZ, a sample of seed of which having beendeposited under NCIMB Accession No. 44097, (b) growing said seed into aharvestable lettuce plant and (c) harvesting lettuce leaves or headsfrom the plant. The invention further comprehends packaging and/orprocessing the lettuce plants, heads or leaves.

Also encompassed by the invention is a container which may comprise oneor more lettuce plants of the invention for harvest of leaves.

Further encompassed by the invention is a method of determining thegenotype of a plant of lettuce variety 45-IC3284 RZ, a sample of seed ofwhich has been deposited under NCIMB Accession No. 44097, or a firstgeneration progeny thereof, which may comprise obtaining a sample ofnucleic acids from said plant and comparing said nucleic acids to asample of nucleic acids obtained from a reference plant, and detecting aplurality of polymorphisms between the two nucleic acid samples, whereinthe plurality of polymorphisms are indicative of lettuce variety45-IC3284 RZ and/or give rise to the expression of any one or more, orall, of the physiological and morphological characteristics of lettucevariety 45-IC3284 RZ of the invention.

Accordingly, it is an object of the invention to not encompass withinthe invention any previously known product, process of making theproduct, or method of using the product such that Applicants reserve theright and hereby disclose a disclaimer of any previously known product,process, or method. It is further noted that the invention does notintend to encompass within the scope of the invention any product,process, or making of the product or method of using the product, whichdoes not meet the written description and enablement requirements of theUSPTO (35 U.S.C. § 112, first paragraph), such that Applicants reservethe right and hereby disclose a disclaimer of any previously describedproduct, process of making the product, or method of using the product.

It is noted that in this disclosure and particularly in the claims,terms such as “comprises”, “comprised”, and “comprising” and the like(e.g., “includes”, “included”, “including”, “contains”, “contained”,“containing”, “has”, “had”, “having”, etc.) can have the meaningascribed to them in US Patent law, i.e., they are open ended terms. Forexample, any method that “comprises,” “has” or “includes” one or moresteps is not limited to possessing only those one or more steps and alsocovers other unlisted steps. Similarly, any plant that “comprises,”“has” or “includes” one or more traits is not limited to possessing onlythose one or more traits and covers other unlisted traits. Similarly,the terms “consists essentially of” and “consisting essentially of” havethe meaning ascribed to them in US Patent law, e.g., they allow forelements not explicitly recited, but exclude elements that are found inthe prior art or that affect a basic or novel characteristic of theinvention. See also MPEP § 2111.03. In addition, the term “about” isused to indicate that a value includes the standard deviation of errorfor the device or method being employed to determine the value.

These and other embodiments are disclosed or are obvious from andencompassed by the following Detailed Description.

DEPOSIT

The Deposit with NCIMB Ltd, Ferguson Building, Craibstone Estate,Bucksburn, Aberdeen AB21 9YA, UK, on Dec. 12, 2022, under NCIMBAccession number 44097 was made and accepted pursuant to the terms ofthe Budapest Treaty. Upon issuance of a patent, all restrictions uponthe deposit will be removed, and the deposit is intended to meet therequirements of 37 CFR §§ 1.801-1.809. The deposit will be irrevocablyand without restriction or condition released to the public upon theissuance of a patent and for the enforceable life of the patent. Thedeposit will be maintained in the depository for a period of 30 years,or 5 years after the last request, or for the effective life of thepatent, whichever is longer, and will be replaced if necessary duringthat period.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description, given by way of example, but notintended to limit the invention solely to the specific embodimentsdescribed, may best be understood in conjunction with the accompanyingdrawing, in which:

FIG. 1 is an illustration of six different shapes of the fourth leaffrom a 20-day old seedling grown under optimal conditions.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides methods and compositions relating to plants,seeds and derivatives of a new lettuce variety herein referred to aslettuce variety 45-IC3284 RZ. Lettuce variety 45-IC3284 RZ is a uniformand stable line, distinct from other such lines.

In a preferred embodiment, the specific type of breeding method employedfor developing a lettuce cultivar is pedigree selection, where bothsingle plant selection and mass selection practices are employed.Pedigree selection, also known as the “Vilmorin system of selection,” isdescribed in Fehr, W., Principles of Cultivar Development, Volume I,MacMillan Publishing Co., which is hereby incorporated by reference.

When pedigree selection is applied, in general selection is firstpracticed among F₂ plants. In the next season, the most desirable F₃lines are first identified, and then desirable F₃ plants within eachline are selected. The following season and in all subsequentgenerations of inbreeding, the most desirable families are identifiedfirst, then desirable lines within the selected families are chosen, andfinally desirable plants within selected lines are harvestedindividually. A family refers to lines that were derived from plantsselected from the same progeny from the preceding generation.

Using this pedigree method, two parents may be crossed using anemasculated female and a pollen donor (male) to produce F₁ offspring.Lettuce is an obligate self-pollination species, which means that pollenis shed before stigma emergence, assuring 100% self-fertilization.Therefore, in order to optimize crossing, a method of misting may beused to wash the pollen off prior to fertilization to assure crossing orhybridization.

Parental varieties are selected from commercial varieties thatindividually exhibit one or more desired phenotypes. Additionally, anybreeding method involving selection of plants for the desired phenotypemay be used in the method of the present invention.

The F₁ may be self-pollinated to produce a segregating F₂ generation.Individual plants may then be selected which represent the desiredphenotype in each generation (F₃, F₄, F₅, etc.) until the traits arehomozygous or fixed within a breeding population.

A detailed description of the development of lettuce variety 45-IC3284RZ is described in Table 1. The seedlot in year 7, seedlot, wasdeposited with the NCIMB under NCIMB Accession No. 44097.

TABLE 1 Year Description Location 0 Final F1-cross in glasshouseFijnaart, The Netherlands 1 F1 plant grown for F2 seed Fijnaart, TheNetherlands production in glasshouse 1-2 F2 plant selected in open fieldDaylesford, Australia followed by F3 seed production 2-3 F3 plantselected in open field Fijnaart, TheNetherlands followed by F4 seedproduction 3 F4 plant selected in open field Fijnaart, The Netherlandsfollowed by F5 seed production 4-5 F5 plant selected in open fieldFijnaart, The Netherlands followed by F6 seed production 5-6 F6 plantselected in open field Salinas, United States followed by F7 seedproduction 6-7 F7 plant selected in open field Salinas, United Statesfollowed by F8 seed production 7 F8 line established uniform,Daylesford, Australia multiplied in plastic tunnel, seed lot 20R.284.

In one embodiment, a plant of the invention has all the physiologicaland morphological characteristics of lettuce variety 45-IC3284 RZ. Thesecharacteristics of a lettuce plant of the invention, e.g. variety45-IC3284 RZ, are summarized in Table 2.

Next to the physiological and morphological characteristics mentioned inTable 2, a plant of the invention also exhibits resistance to downymildew (Bremia lactucae Regel.) races B1:16EU to B1:37EU and B1:1US toB1:9US, resistance to currant-lettuce aphid (Nasonovia ribisnigri)biotype Nr:0, resistance to Lettuce mosaic virus (LMV), intermediateresistance to Fusarium oxysporum f sp. lactuace race 4 (Fol:4), andresistance to lettuce root aphids pb (Pemphigus bursarius).

As used herein resistance against Bremia lactucae is defined as theability of a plant to resist infection by each of the various strainsB1:16EU to B1:37EU, B1:1US to B1:9US of Bremia lactucae Regel. in allstages between the seedling stage and the harvestable plant stage.B1:16EU to B1:37EU means strains B1:16EU, B1:17EU, B1:18EU, B1:20EU,B1:21EU, B1:22EU, B1:23EU, B1:24EU, B1:25EU, B1:26EU, B1:27EU, B1:29EU,B1:30EU, B1:31EU, B1:32EU, B1:33EU, B1:34EU, B1:35EU, B1:36EU, B1:37EU(Van Ettekoven K, Van der Arend A J M, 1999. identification anddenomination of ‘new’ races of Bremia lactucae. In: Lebeda A, KristkovaE (eds.) Eucarpia leafy vegetables '99. Palacky University, Olomouc,Czech Republic, 1999: 171-175; Van der Arend, A. J. M., Gautier, J.,Guenard, M., Michel, H., Moreau, B., de Ruijter, J., Schut, J. W. and deWitte, I. (2003). Identification and denomination of ‘new’ races ofBremia lactucae in Europe by IBEB until 2002. In: Eucarpia leafyvegetables 2003. Proceedings of the Eucarpia Meeting on leafy vegetablesgenetics and breeding. Noorwijkerhout, The Netherlands. Eds. Van HintumT., Lebeda A., Pink D., Schut J. pp 151-160; Van der Arend A J M,Gautier J, Grimault V, Kraan P, Van der Laan R, Mazet J, Michel H, SchutJ W, Smilde D, De Witte I (2006) Identification and denomination of“new” races of Bremia lactucae in Europe by IBEB until 2006;incorporated herein by reference; Plantum NL (Dutch association forbreeding, tissue culture, production and trade of seeds and youngplants), IBEB press release, “New race of Bremia lactucae B1:27identified and nominated”, May 2010; Plantum NL, IBEB press release,“New races of Bremia lactucae, B1:29, B1:30 and B1:31 identified andnominated”, August 2013; Plantum NL, IBEB press release, “A new race ofBremia lactucae, B1:32 identified and nominated in Europe”, May 2015;Plantum NL, IBEB-EU press release, “A new race of Bremia lactucae,B1:33EU identified and denominated in Europe”, May 2017; Plantum NL,IBEB-EU press release, “Two new races of Bremia lactucae, B1:34EU andB1:35EU identified and nominated in Europe”, July 2018; Plantum NL,IBEB-EU press release, “A new race of Bremia lactucae, B1:36EUidentified and denominated in Europe”, July 2019; Plantum NL, IBEB EUpress release, “A new race of Bremia lactuceae, B1:37EU identified anddenominated in Europe, June 2021). B1:1US to B1:9US means B1:1US,B1:2US, B1:3US, B1:4US, B1:5US, B1:6US, B1:7US, B1:8US, B1:9US(Schettini, T. M., Legg, E. J., Michelmore, R. W., 1991. Insensitivityto metalaxyl in California populations of Bremia lactucae and resistanceof California lettuce cultivars to downy mildew, Phytopathology 81(1).p. 64-′70; Michelmore R. & Ochoa. O. “Breeding Crisphead Lettuce.” In:California Lettuce Research Board, Annual Report 2005-2006, 2006,Salinas, Calif., pp. 55-68; http://bremia.ucdavis.edu/data/B1_9US.pdf).

Resistance typically is tested by two interchangeable methods, describedby Bonnier, F. J. M. et al. (Euphytica, 61(3):203-211, 1992;incorporated herein by reference). One method involves inoculating 7-dayold seedlings, and observing sporulation 10 to 14 days later. The othermethod involves inoculating leaf discs with a diameter of 18 mm obtainedfrom a non-senescent, fully grown true leaf and observing sporulation 10days later.

As used herein, resistance against Nasonovia ribisnigri (Mosley), orcurrant-lettuce aphid, is defined as the plant characteristic whichresults in a non-feeding response of the aphid on the leaves of theplant in all stages between 5 true-leaf stage and harvestable plantstage (U.S. Pat. No. 5,977,443 to Jansen, J. P. A., “Aphid Resistance inComposites,” p. 12, 1999; incorporated herein by reference).

Resistance is tested by spreading at least ten aphids of biotype Nr:0 ona plant in a plant stage between 5 true leaves and harvestable stage,and observing the density of the aphid population on the plant as wellas the growth reduction after 14 days in a greenhouse, with temperaturesettings of 23 degrees Celsius in daytime and 21 degrees Celsius atnight. Day length is kept at 18 hours by assimilation lights.

As used herein, resistance against lettuce mosaic virus (LMV) may bedefined as the ability of the plant to grow normally after LMV infectionand to inhibit the virus transmission to seed. Resistance may be testedby mechanical inoculation of young plants in a climate cell orgreenhouse, as described by Pink, D. A. C. et al. (Plant Pathology,41(1): 5-12, 1992), incorporated herein by reference. Inoculatedresistant plants grow just as well as uninoculated plants and show nochlorosis or mosaic symptoms. The LMV isolate, which may be used fortesting, is Ls-1 (International Union for the Protection of NewVarieties of Plants [UPOV], Guidelines for the conduct of tests fordistinctness, uniformity and stability; 30 lettuce (Lactuca sativa L.),2002, p. 35; incorporated herein by reference).

As used herein, resistance against Fusarium oxysporum f.sp. lactucae, isdefined as the ability of a plant to resist infection by races Fol:4,also known as race 4. In 2015 a new variant of Fusarium in lettuce,Fusarium oxysporum f. sp. lactucae race 4, was identified and describedby Gilardi et al. (2017). ISF has proposed a new differential set totheir Disease Resistance Terminology Working Group (DRT WG) todifferentiate this new race.https://worldseed.org/wp-content/uploads/2022/05/Heads-UP-2022_Differential_sets_Fol_race4.pdf.After testing several isolates by all breeding companies active withlettuce, the isolate collected in the Netherlands and published byGilardi et al., 2017 (a new race of Fusarium oxysporum f. sp. lactucaeof lettuce. Plant Pathol. 66, 677-688. Doi 10. 1111/ppa.12616) waschosen as a reference isolate. The test protocol was used as describedin https://worldseed.org/document/isf-fnal-report-fol-race-4-lettuce/.

As used herein, resistance to Pemphigus bursarius, or root aphid, isdefined as the ability of a plant to resist infection to root aphids.Resistance may be tested by the method described by Ellis et al., (1994,Annals of Applied Biology 124:141-151; incorporated herein byreference).

Embodiments of the inventions advantageously have one or more, and mostadvantageously all, of these characteristics.

In the below Table 2, the characteristics of “45-IC3284 RZ” are shown.

TABLE 2 SEED Seed Color Black PLANT Plant: Head Diameter Medium to largePlant: Degree of Overlapping of Strong Upper Part of Leaves LEAF Leaf:Leaf Attitude Semi-erect Leaf: Number of Divisions Absent or very fewLeaf: Shape Medium oblate Leaf: Shape of Apex Rounded Leaf: LongitudinalSection Flat Leaf: Anthocyanin Coloration Absent or very weak Leaf:Color Green Leaf: Intensity of Green Color Medium Leaf: Glossiness ofUpper Side Weak Leaf Thickness Thick Leaf: Blistering Medium Leaf: Sizeof Blisters small Leaf: Undulation of Margin Weak to medium Leaf: Typeof Incisions of Margin Irregularly dentate Leaf: Depth of Incisions ofMargin Shallow to medium Leaf: Depth of Secondary Incisions of shallowMargin Leaf: Density of Incisions of Margin Sparse to medium Leaf:Venation flabellate HEAD Head: Size Medium to large Head: Shape inLongitudinal Section Circular Head: Density Dense to very dense HARVESTTime of Harvest Maturity Medium to late BOLTING Bolting: Time ofBeginning of Bolting Very late Auxilliary Sprouting Absent or weakBolting: Bolting Stem: Fasciation Absent or very weak PLANT TYPE PlantType Iceberg COTYLEDON TO FOURTH LEAF STAGE Shape of CotyledonsIntermediate Shape of Fourth Leaf Elongated PLANT Plant head Diameter 16Plant Spread of Frame Leaves 52 Plant Number of Leaves — LEAF Leaf:Number of Divisions  1 BUTT Butt: Shape Rounded Butt: Midrib Moderatelyraised HARVEST MATURITY Time of harvest Maturity (Days) 97

In one aspect the invention provides a new type of lettuce (Lactucasativa) variety, designated 45-IC3284 RZ. Lettuce cultivar 45-IC3284 RZexhibits a combination of traits including resistance to downy mildew(Bremia lactucae) races B1:16EU to B1:37EU and B1:1US to B1:9US,resistance to currant-lettuce aphid (Nasonovia ribisnigri) biotype Nr:0,resistance to Lettuce mosaic virus (LMV), intermediate resistance toFusarium oxysporum f sp. lactuace race 4 (Fol:4), resistance to lettuceroot aphids pb (Pemphigus bursarius), as well as having mature leaveswith medium blistering.

In an embodiment, the invention relates to lettuce plants that have allthe physiological and morphological characteristics of the invention andhave acquired said characteristics by introduction of the geneticinformation that is responsible for the characteristics from a suitablesource, either by conventional breeding, or genetic modification, inparticular by cisgenesis or transgenesis. Cisgenesis is geneticmodification of plants with a natural gene, coding for an (agricultural)trait, from the crop plant itself or from a sexually compatible donorplant. Transgenesis is genetic modification of a plant with a gene froma non-crossable species or a synthetic gene.

Just as useful traits that may be introduced by backcrossing, usefultraits may be introduced directly into the plant of the invention, beinga plant of lettuce variety 45-IC3284 RZ, by genetic transformationtechniques; and, such plants of lettuce variety 45-IC3284 RZ that haveadditional genetic information introduced into the genome or thatexpress additional traits by having the DNA coding there for introducedinto the genome via transformation techniques, are within the ambit ofthe invention, as well as uses of such plants, and the making of suchplants.

Genetic transformation may therefore be used to insert a selectedtransgene into the plant of the invention, being a plant of lettucevariety 45-IC3284 RZ or may, alternatively, be used for the preparationof transgenes which may be introduced by backcrossing. Methods for thetransformation of plants, including lettuce, are well known to those ofskill in the art.

Vectors used for the transformation of lettuce cells are not limited solong as the vector may express an inserted DNA in the cells. Forexample, vectors which may comprise promoters for constitutive geneexpression in lettuce cells (e.g., cauliflower mosaic virus 35Spromoter) and promoters inducible by exogenous stimuli may be used.Examples of suitable vectors include pBI binary vector. The “lettucecell” into which the vector is to be introduced includes various formsof lettuce cells, such as cultured cell suspensions, protoplasts, leafsections, and callus. A vector may be introduced into lettuce cells byknown methods, such as the polyethylene glycol method, polycationmethod, electroporation, Agrobacterium-mediated transfer, particlebombardment and direct DNA uptake by protoplasts. To effecttransformation by electroporation, one may employ either friabletissues, such as a suspension culture of cells or embryogenic callus oralternatively one may transform immature embryos or other organizedtissue directly. In this technique, one would partially degrade the cellwalls of the chosen cells by exposing them to pectin-degrading enzymes(pectolyases) or mechanically wound tissues in a controlled manner.

A particularly efficient method for delivering transforming DNA segmentsto plant cells is microprojectile bombardment. In this method, particlesare coated with nucleic acids and delivered into cells by a propellingforce. Exemplary particles include those which may be comprised oftungsten, platinum, and preferably, gold. For the bombardment, cells insuspension are concentrated on filters or solid culture medium.Alternatively, immature embryos or other target cells may be arranged onsolid culture medium. The cells to be bombarded are positioned at anappropriate distance below the macroprojectile stopping plate. Anillustrative embodiment of a method for delivering DNA into plant cellsby acceleration is the Biolistics Particle Delivery System, which may beused to propel particles coated with DNA or cells through a screen, suchas a stainless steel or Nytex screen, onto a surface covered with targetlettuce cells. The screen disperses the particles so that they are notdelivered to the recipient cells in large aggregates. It is believedthat a screen intervening between the projectile apparatus and the cellsto be bombarded reduces the size of projectiles aggregate and maycontribute to a higher frequency of transformation by reducing thedamage inflicted on the recipient cells by projectiles that are toolarge. Microprojectile bombardment techniques are widely applicable, andmay be used to transform virtually any plant species, including a plantof lettuce variety 45-IC3284 RZ.

Agrobacterium-mediated transfer is another widely applicable system forintroducing gene loci into plant cells. An advantage of the technique isthat DNA may be introduced into whole plant tissues, thereby bypassingthe need for regeneration of an intact plant from a protoplast.Agrobacterium transformation vectors are capable of replication in E.coli as well as Agrobacterium, allowing for convenient manipulations.Moreover, advances in vectors for Agrobacterium-mediated gene transferhave improved the arrangement of genes and restriction sites in thevectors to facilitate the construction of vectors capable of expressingvarious polypeptide coding genes. The vectors have convenientmulti-linker regions flanked by a promoter and a polyadenylation sitefor direct expression of inserted polypeptide coding genes.Additionally, Agrobacterium containing both armed and disarmed Ti genesmay be used for transformation. In those plant strains whereAgrobacterium-mediated transformation is efficient, it is the method ofchoice because of the facile and defined nature of the gene locustransfer. The use of Agrobacterium-mediated plant integrating vectors tointroduce DNA into plant cells, including lettuce plant cells, is wellknown in the art (See, e.g. U.S. Pat. Nos. 7,250,560 and 5,563,055).

Transformation of plant protoplasts also may be achieved using methodsbased on calcium phosphate precipitation, polyethylene glycol treatment,electroporation, and combinations of these treatments.

A number of promoters have utility for plant gene expression for anygene of interest including but not limited to selectable markers,scoreable markers, genes for pest tolerance, disease resistance,nutritional enhancements and any other gene of agronomic interest.Examples of constitutive promoters useful for lettuce plant geneexpression include, but are not limited to, the cauliflower mosaic virus(CaMV) P-35S promoter, a tandemly duplicated version of the CaMV 35Spromoter, the enhanced 35S promoter (P-e35 S), the nopaline synthasepromoter, the octopine synthase promoter, the figwort mosaic virus(P-FMV) promoter (see U.S. Pat. No. 5,378,619), an enhanced version ofthe FMV promoter (P-eFMV) where the promoter sequence of P-FMV isduplicated in tandem, the cauliflower mosaic virus 19S promoter, asugarcane bacilliform virus promoter, a commelina yellow mottle viruspromoter, the promoter for the thylakoid membrane proteins (psaD, psaF,psaE, PC, FNR, atpC, atpD, cab, rbcS) (see U.S. Pat. No. 7,161,061), theCAB-1 promoter (see U.S. Pat. No. 7,663,027), the promoter from maizeprolamin seed storage protein (see U.S. Pat. No. 7,119,255), and otherplant DNA virus promoters known to express in plant cells. A variety ofplant gene promoters that are regulated in response to environmental,hormonal, chemical, and/or developmental signals may be used forexpression of an operably linked gene in plant cells, includingpromoters regulated by (1) heat, (2) light (e.g., pea rbcS-3A promoter,maize rbcS promoter, or chlorophyll a/b-binding protein promoter), (3)hormones, such as abscisic acid, (4) wounding (e.g., wunl, or (5)chemicals such as methyl jasmonate, salicylic acid, or Safener. It mayalso be advantageous to employ organ-specific promoters.

Exemplary nucleic acids which may be introduced to the lettuce varietyof this invention include, for example, DNA sequences or genes fromanother species, or even genes or sequences which originate with or arepresent in lettuce species, but are incorporated into recipient cells bygenetic engineering methods rather than classical reproduction orbreeding techniques. However, the term “exogenous” is also intended torefer to genes that are not normally present in the cell beingtransformed, or perhaps simply not present in the form, structure, etc.,as found in the transforming DNA segment or gene, or genes which arenormally present and that one desires to express in a manner thatdiffers from the natural expression pattern, e.g., to over-express.Thus, the term “exogenous” gene or DNA is intended to refer to any geneor DNA segment that is introduced into a recipient cell, regardless ofwhether a similar gene may already be present in such a cell. The typeof DNA included in the exogenous DNA may include DNA which is alreadypresent in the plant cell, DNA from another plant, DNA from a differentorganism, or a DNA generated externally, such as a DNA sequencecontaining an antisense message of a gene, or a DNA sequence encoding asynthetic or modified version of a gene.

Many hundreds if not thousands of different genes are known and couldpotentially be introduced into a plant of lettuce variety 45-IC3284 RZ.Non-limiting examples of particular genes and corresponding phenotypesone may choose to introduce into a lettuce plant include one or moregenes for insect tolerance, pest tolerance such as genes for fungaldisease control, herbicide tolerance, and genes for quality improvementssuch as yield, nutritional enhancements, environmental or stresstolerances, or any desirable changes in plant physiology, growth,development, morphology or plant product(s).

Alternatively, the DNA coding sequences may affect these phenotypes byencoding a non-translatable RNA molecule that causes the targetedinhibition of expression of an endogenous gene, for example viaantisense- or cosuppression-mediated mechanisms. The RNA could also be acatalytic RNA molecule (i.e., a ribozyme) engineered to cleave a desiredendogenous mRNA product. Thus, any gene which produces a protein or mRNAwhich expresses a phenotype or morphology change of interest is usefulfor the practice of the present invention (See also U.S. Pat. No.7,576,262, “Modified gene-silencing RNA and uses thereof.”).

U.S. Pat. Nos. 7,230,158, 7,122,720, 7,081,363, 6,734,341, 6,503,732,6,392,121, 6,087,560, 5,981,181, 5,977,060, 5,608,146, 5,516,667, eachof which, and all documents cited therein are hereby incorporated hereinby reference, consistent with the above INCORPORATION BY REFERENCEsection, are additionally cited as examples of U.S. patents that mayconcern transformed lettuce and/or methods of transforming lettuce orlettuce plant cells, and techniques from these US patents, as well aspromoters, vectors, etc., may be employed in the practice of thisinvention to introduce exogenous nucleic acid sequence(s) into a plantof lettuce variety 45-IC3284 RZ (or cells thereof), and exemplify someexogenous nucleic acid sequence(s) which may be introduced into a plantof lettuce variety 45-IC3284 RZ (or cells thereof) of the invention, aswell as techniques, promoters, vectors etc., to thereby obtain furtherplants of lettuce variety 45-IC3284 RZ, plant parts and cells, seeds,other propagation material harvestable parts of these plants, etc. ofthe invention, e.g. tissue culture, including a cell or protoplast, suchas an embryo, meristem, cotyledon, pollen, leaf, anther, root, root tip,pistil, flower, seed or stalk.

The invention further relates to propagation material for producingplants of the invention. Such propagation material may comprise interalia seeds of the claimed plant and parts of the plant that are involvedin sexual reproduction. Such parts are for example selected from thegroup consisting of seeds, microspores, pollen, ovaries, ovules, embryosacs and egg cells. In addition, the invention relates to propagationmaterial which may comprise parts of the plant that are suitable forvegetative reproduction, for example cuttings, roots, stems, cells,protoplasts.

According to a further aspect thereof the propagation material of theinvention may comprise a tissue culture of the claimed plant. The tissueculture may comprise regenerable cells. Such tissue culture may bederived from leaves, pollen, embryos, cotyledon, hypocotyls,meristematic cells, roots, root tips, anthers, flowers, seeds and stems.(See generally U.S. Pat. No. 7,041,876 on lettuce being recognized as aplant that may be regenerated from cultured cells or tissue).

Also, the invention comprehends methods for producing a seed of a“45-IC3284 RZ”-derived lettuce plant which may comprise (a) crossing aplant of lettuce variety 45-IC3284 RZ, a sample of seed of which havingbeen deposited under NCIMB Accession No. 44097, with a second lettuceplant, and (b) whereby seed of a 45-IC3284 RZ-derived lettuce plantform. Such a method may further comprise (c) crossing a plant grown from45-IC3284 RZ-derived lettuce seed with itself or with a second lettuceplant to yield additional 45-IC3284 RZ-derived lettuce seed, (d) growingthe additional 45-IC3284 RZ-derived lettuce seed of step (c) to yieldadditional 45-IC3284 RZ-derived lettuce plants, and (e) repeating thecrossing and growing of steps (c) and (d) for an additional 3-10generations to further generate 45-IC3284 RZ-derived lettuce plants.

The invention further relates to the above methods that may furthercomprise selecting at steps b), d), and e), a 45-IC3284 RZ-derivedlettuce plant, exhibiting one or more or all of the physiological andmorphological characteristics of lettuce variety 45-IC3284 RZ, a sampleof seed of said variety having been deposited under NCIMB Accession No.44097, and other selected traits.

In particular, the invention relates to methods for producing a seed ofa 45-IC3284 RZ-derived lettuce plant which may comprise (a) crossing aplant of lettuce variety 45-IC3284 RZ, a sample of seed of which havingbeen deposited under NCIMB Accession No. 44097, with a second lettuceplant and (b) whereby seed of a 45-IC3284 RZ-derived lettuce plantforms, wherein such a method may further comprise (c) crossing a plantgrown from 45-IC3284 RZ-derived lettuce seed with itself or with asecond lettuce plant to yield additional 45-IC3284 RZ-derived lettuceseed, (d) growing the additional 45-IC3284 RZ-derived lettuce seed ofstep (c) to yield additional 45-IC3284 RZ-derived lettuce plants andselecting plants exhibiting one or more or all of the physiological andmorphological characteristics of lettuce variety 45-IC3284 RZ, and (e)repeating the crossing and growing of steps (c) and (d) for anadditional 3-10 generations to further generate 45-IC3284 RZ-derivedlettuce plants that exhibit one or more or all of the physiological andmorphological characteristics of lettuce variety 45-IC3284 RZ.

The invention additionally provides a method of introducing at least onenew trait into a plant of lettuce variety 45-IC3284 RZ which maycomprise: (a) crossing a plant of lettuce variety 45-IC3284 RZ, a sampleof seed of which having been deposited under NCIMB Accession No. 44097,with a second lettuce plant that may comprise at least one new trait toproduce progeny seed; (b) harvesting and planting the progeny seed toproduce at least one progeny plant of a subsequent generation, whereinthe progeny plant may comprise the at least one new trait; (c) crossingthe selected progeny plant with a plant of lettuce variety 45-IC3284 RZ,to produce backcross progeny seed; (d) harvesting and planting thebackcross progeny seed to produce a backcross progeny plant, (e)repeating steps (c) and (d) for at least three additional generations toproduce backcross progeny that may comprise the at least one new traitand all of the physiological and morphological characteristics of aplant of lettuce variety 45-IC3284 RZ, when grown in the sameenvironmental conditions. The invention, of course, includes a lettuceplant produced by this method.

Backcrossing may also be used to improve an inbred plant. Backcrossingtransfers a specific desirable trait from one inbred or non-inbredsource to an inbred that lacks that trait. This may be accomplished, forexample, by first crossing a superior inbred (A) (recurrent parent) to adonor inbred (non-recurrent parent), which carries the appropriate locusor loci for the trait in question. The progeny of this cross are thenmated back to the superior recurrent parent (A) followed by selection inthe resultant progeny for the desired trait to be transferred from thenon-recurrent parent. After five or more backcross generations withselection for the desired trait, the progeny are heterozygous for locicontrolling the characteristic being transferred, but are like thesuperior parent for most or almost all other loci. The last backcrossgeneration would be selfed to give pure breeding progeny for the traitbeing transferred. When a plant of lettuce variety 45-IC3284 RZ, asample of seed of which having been deposited under NCIMB Accession No.44097, is used in backcrossing, offspring retaining one or more or allof the physiological and morphological characteristics of lettucevariety 45-IC3284 RZ are progeny within the ambit of the invention.Backcrossing methods may be used with the present invention to improveor introduce a characteristic into a plant of the invention, being aplant of lettuce variety 45-IC3284 RZ. See, e.g., U.S. Pat. No.7,705,206 (incorporated herein by reference consistent with the aboveINCORPORATION BY REFERENCE section), for a general discussion relatingto backcrossing.

The invention further involves a method of determining the genotype of aplant of lettuce variety 45-IC3284 RZ, a sample of seed of which hasbeen deposited under NCIMB Accession No. 44097, or a first generationprogeny thereof, which may comprise obtaining a sample of nucleic acidsfrom said plant and detecting in said nucleic acids a plurality ofpolymorphisms. This method may additionally comprise the step of storingthe results of detecting the plurality of polymorphisms on a computerreadable medium. The plurality of polymorphisms are indicative of and/orgive rise to the expression of the physiological and morphologicalcharacteristics of lettuce variety 45-IC3284 RZ.

There are various ways of obtaining genotype data from a nucleic acidsample. Genotype data may be gathered which is specific for certainphenotypic traits (e.g. gene sequences), but also patterns of randomgenetic variation may be obtained to construct a so-called DNAfingerprint. Depending on the technique used a fingerprint may beobtained that is unique for lettuce variety 45-IC3284 RZ. Obtaining aunique DNA fingerprint depends on the genetic variation present in avariety and the sensitivity of the fingerprinting technique. A techniqueknown in the art to provide a good fingerprint profile is called AFLPfingerprinting technique (See generally U.S. Pat. No. 5,874,215), butthere are many other marker based techniques, such as RFLP (orRestriction fragment length polymorphism), SSLP (or Simple sequencelength polymorphism), RAPD (or Random amplification of polymorphic DNA)VNTR (or Variable number tandem repeat), Microsatellite polymorphism,SSR (or Simple sequence repeat), STR (or Short tandem repeat), SFP (orSingle feature polymorphism) DArT (or Diversity Arrays Technology), RADmarkers (or Restriction site associated DNA markers) (e.g. Baird et al.PloS One Vol. 3 e3376, 2008; Semagn et al. African Journal ofBiotechnology Vol. 5 number 25 pp. 2540-2568, 29 December, 2006).Nowadays, sequence-based methods are utilizing Single NucleotidePolymorphisms (SNPs) that are randomly distributed across genomes, as acommon tool for genotyping (e.g. Elshire et al. PloS One Vol. 6: e19379,2011; Poland et al. PloS One Vol. 7: e32253; Truong et al. PLoS One Vol.7 number 5: e37565, 2012).

With any of the aforementioned genotyping techniques, polymorphisms maybe detected when the genotype and/or sequence of the plant of interestis compared to the genotype and/or sequence of one or more referenceplants. As used herein, the genotype and/or sequence of a referenceplant may be derived from, but is not limited to, any one of thefollowing: parental lines, closely related plant varieties or species,complete genome sequence of a related plant variety or species, or thede novo assembled genome sequence of one or more related plant varietiesor species. For example, it is possible to detect polymorphisms for thecharacteristic of resistance to lettuce root aphids pb by comparing thegenotype and/or the sequence of lettuce variety 45-IC3284 RZ with thegenotype and/or the sequence of one or more reference plants. Thereference plant(s) used for comparison in this example may for examplebe, but is not limited to, the comparison variety Regnas RZ.

The polymorphism revealed by these techniques may be used to establishlinks between genotype and phenotype. The polymorphisms may thus be usedto predict or identify certain phenotypic characteristics, individuals,or even species. The polymorphisms are generally called markers. It iscommon practice for the skilled artisan to apply molecular DNAtechniques for generating polymorphisms and creating markers.

The polymorphisms of this invention may be provided in a variety ofmediums to facilitate use, e.g. a database or computer readable medium,which may also contain descriptive annotations in a form that allows askilled artisan to examine or query the polymorphisms and obtain usefulinformation.

As used herein “database” refers to any representation of retrievablecollected data including computer files such as text files, databasefiles, spreadsheet files and image files, printed tabulations andgraphical representations and combinations of digital and image datacollections. In a preferred aspect of the invention, “database” refersto a memory system that may store computer searchable information.

As used herein, “computer readable media” refers to any medium that maybe read and accessed directly by a computer. Such media include, but arenot limited to: magnetic storage media, such as floppy discs, hard disc,storage medium and magnetic tape; optical storage media such as CD-ROM;electrical storage media such as RAM, DRAM, SRAM, SDRAM, ROM; and PROMs(EPROM, EEPROM, Flash EPROM), and hybrids of these categories such asmagnetic/optical storage media. A skilled artisan may readily appreciatehow any of the presently known computer readable mediums may be used tocreate a manufacture which may comprise computer readable medium havingrecorded thereon a polymorphism of the present invention.

As used herein, “recorded” refers to the result of a process for storinginformation in a retrievable database or computer readable medium. Forinstance, a skilled artisan may readily adopt any of the presently knownmethods for recording information on computer readable medium togenerate media which may comprise the polymorphisms of the presentinvention. A variety of data storage structures are available to askilled artisan for creating a computer readable medium where the choiceof the data storage structure will generally be based on the meanschosen to access the stored information. In addition, a variety of dataprocessor programs and formats may be used to store the polymorphisms ofthe present invention on computer readable medium.

The present invention further provides systems, particularlycomputer-based systems, which contain the polymorphisms describedherein. Such systems are designed to identify the polymorphisms of thisinvention. As used herein, “a computer-based system” refers to thehardware, software and memory used to analyze the polymorphisms. Askilled artisan may readily appreciate that any one of the currentlyavailable computer-based system are suitable for use in the presentinvention.

Lettuce leaves are sold in packaged form, including without limitationas pre-packaged lettuce salad or as lettuce heads. Mention is made ofU.S. Pat. No. 5,523,136, incorporated herein by reference consistentwith the above INCORPORATION BY REFERENCE section, which providespackaging film, and packages from such packaging film, including suchpackaging containing leafy produce, and methods for making and usingsuch packaging film and packages, which are suitable for use with thelettuce leaves of the invention. Thus, the invention comprehends the useof and methods for making and using the leaves of the lettuce plant ofthe invention, as well as leaves of lettuce plants derived from theinvention.

The invention further relates to a container which may comprise one ormore plants of the invention, or one or more lettuce plants derived froma plant of the invention, in a growth substrate for harvest of leavesfrom the plant in a domestic environment. This way the consumer may pickvery fresh leaves for use in salads. More generally, the inventionincludes one or more plants of the invention or one or more plantsderived from lettuce of the invention, wherein the plant is in aready-to-harvest condition, including with the consumer picking his own,and further including a container which may comprise one or more ofthese plants.

The invention is further described by the following numbered paragraphs:

1. A seed of lettuce variety 45-IC3284 RZ, a sample of seed of saidvariety having been deposited under NCIMB Accession No. 44097.

2. A lettuce plant grown from the seed of paragraph 1.

3. A lettuce plant, or a part thereof, having all the physiological andmorphological characteristics of the lettuce plant of paragraph 2.

4. A part of the lettuce plant of paragraph 2, wherein said partcomprises a microspore, pollen, ovary, ovule, embryo sac, egg cell,cutting, root, stem, cell or protoplast.

5. A tissue culture of regenerable cells or protoplasts from the lettuceplant of paragraph 2.

6. The tissue culture as in paragraph 6, wherein said cells orprotoplasts of the tissue culture are derived from a tissue comprising aleaf, pollen, embryo, cotyledon, hypocotyl, meristematic cell, root,root tip, anther, flower, seed or stem.

7. A lettuce plant regenerated from the tissue culture of paragraph 5 or6, wherein the regenerated plant expresses all of the physiological andmorphological characteristics of lettuce variety 45-IC3284 RZ, a sampleof seed of said variety having been deposited under NCIMB Accession No.44097.

8. A method of vegetatively propagating a plant of lettuce variety45-IC3284 RZ comprising (a) collecting tissue capable of beingpropagated from a lettuce plant of paragraph 2, (b) cultivating thetissue to obtain proliferated shoots and rooting the proliferated shootsto obtain rooted plantlets, and (c) optionally growing plants from therooted plantlets.

9. A method for producing a progeny plant of lettuce cultivar 45-IC3284RZ, comprising crossing a lettuce plant of paragraph 2 with itself orwith another lettuce plant, harvesting the resultant seed, and growingsaid seed.

10. A progeny plant produced by the method of paragraph 9, wherein saidprogeny exhibits all the morphological and physiological characteristicsof the lettuce variety designated 45-IC3284 RZ, a sample of seed of saidvariety having been deposited under NCIMB Accession No. 44097.

11. The progeny plant as in paragraph 10, wherein said progeny plant ismodified in one or more other characteristics.

12. A lettuce plant of paragraph 2 further comprising a transgene.

13. The plant as in paragraph 12, wherein the transgene is introducedvia transformation.

14. A method for producing a modified lettuce plant comprisingmutagenizing the seed of paragraph 1 and growing said seed.

15. A method for producing a modified lettuce plant comprisingmutagenizing the plant of any one of paragraphs 2, 3 or 7.

16. A method for producing a modified lettuce plant comprisingmutagenizing the part of plant of any one of paragraphs 3 or 4.

17. A method for producing a modified lettuce plant comprisingmutagenizing the tissue culture of any one of paragraphs 5 or 6.

18. A method of producing a lettuce seed comprising crossing a maleparent lettuce plant with a female parent lettuce plant and harvestingthe resultant lettuce seed, wherein said male parent lettuce plant orsaid female parent lettuce plant is the lettuce plant of paragraph 2.

19. An F1 lettuce seed produced by the method of paragraph 18.

20. A lettuce plant produced by growing the seed of paragraph 19.

21. A method for producing a seed of a 45-IC3284 RZ-derived lettuceplant comprising (a) crossing a plant of lettuce variety 45-IC3284 RZ, asample of seed of which having been deposited under NCIMB Accession No.44097, with a second lettuce plant, and (b) whereby seed of a 45-IC3284RZ-derived lettuce plant forms.

22. The method of paragraph 21 further comprising (c) crossing a plantgrown from 45-IC3284 RZ-derived lettuce seed with itself or with asecond lettuce plant to yield additional 45-IC3284 RZ-derived lettuceseed, (d) growing the additional 45-IC3284 RZ-derived lettuce seed ofstep (c) to yield additional 45-IC3284 RZ-derived lettuce plants, and(e) repeating the crossing and growing of steps (c) and (d) for anadditional 3-10 generations to generate further 45-IC3284 RZ-derivedlettuce plants, and (f) whereby seed of a 45-IC3284 RZ-derived lettuceplant forms.

23. A method of introducing at least one new trait into a plant oflettuce variety 45-IC3284 RZ comprising: (a) crossing a plant of lettucevariety 45-IC3284 RZ, a sample of seed of which having been depositedunder NCIMB Accession No. 44097, with a second lettuce plant thatcomprises at least one new trait to produce progeny seed, (b) harvestingand planting the progeny seed to produce at least one progeny plant of asubsequent generation, wherein the progeny plant comprises the at leastone new trait, (c) crossing the progeny plant with a plant of lettucevariety 45-IC3284 RZ to produce backcross progeny seed, (d) harvestingand planting the backcross progeny seed to produce a backcross progenyplant, and (e) repeating steps (c) and (d) for at least three additionalgenerations to produce a lettuce plant of variety 45-IC3284 RZcomprising at least one new trait and all of the physiological andmorphological characteristics of a plant of lettuce variety 45-IC3284RZ, when grown in the same environmental conditions.

24. The lettuce plant produced by the method of paragraph 23, whereinthe plant comprises the at least one new trait and otherwise all of thephysiological and morphological characteristics of a lettuce plant oflettuce variety 45-IC3284 RZ.

25. A method for producing lettuce leaves as a food product comprisingsowing the seed of paragraph 1 and growing the seed into a harvestablelettuce plant and harvesting the head or leaves of said plant,optionally processing and/or packaging the head or the leaves.

26. A container comprising one or more lettuce plants of paragraph 2 forharvest of leaves.

Having thus described in detail preferred embodiments of the presentinvention, it is to be understood that the invention is not to belimited to particular details set forth in the above description as manyapparent variations thereof are possible without departing from thespirit or scope of the present invention.

What is claimed is:
 1. A seed of lettuce variety 45-IC3284 RZ, a sampleof seed of said variety having been deposited under NCIMB Accession No.44097.
 2. A lettuce plant grown from the seed of claim
 1. 3. A lettuceplant, or a part thereof, having all the physiological and morphologicalcharacteristics of the lettuce plant of claim
 2. 4. A part of thelettuce plant of claim 2, wherein said part comprises a leaf, cotyledon,hypocotyl, meristematic cell, microspore, pollen, ovary, ovule, embryo,embryo sac, egg cell, cutting, root, root tip, anther, flower, stem,cell, seed or protoplast.
 5. A tissue culture of regenerable cells orprotoplasts from the lettuce plant of claim
 2. 6. A lettuce plantregenerated from the tissue culture of claim 5, wherein the regeneratedplant expresses all of the physiological and morphologicalcharacteristics of lettuce variety 45-IC3284 RZ, a sample of seed ofsaid variety having been deposited under NCIMB Accession No.
 44097. 7. Amethod of vegetatively propagating a plant of lettuce variety 45-IC3284RZ, a sample of seed of said variety having been deposited under NCIMBAccession No. 44097, comprising (a) collecting tissue capable of beingpropagated from a lettuce plant of claim 2, (b) cultivating the tissueto obtain proliferated shoots and rooting the proliferated shoots toobtain rooted plantlets, and (c) optionally growing plants from therooted plantlets.
 8. A method for producing a progeny plant of lettucecultivar 45-IC3284 RZ, comprising crossing a lettuce plant of claim 2with itself or with another lettuce plant, harvesting the resultantseed, and growing said seed.
 9. A progeny plant produced by the methodof claim 8, wherein said progeny exhibits all the morphological andphysiological characteristics of the lettuce variety designated45-IC3284 RZ, a sample of seed of said variety having been depositedunder NCIMB Accession No.
 44097. 10. The progeny plant as claimed inclaim 9, wherein said progeny plant is modified in one or more othercharacteristics.
 11. A lettuce plant of claim 2 further comprising atransgene.
 12. The plant as claimed in claim 11, wherein the transgeneis introduced via transformation.
 13. A method for producing a modifiedlettuce plant comprising mutagenizing the seed of claim 1 and growingsaid seed.
 14. A method for producing a modified lettuce plantcomprising mutagenizing the plant of any one of claim 2, 3 or
 6. 15. Amethod for producing a modified lettuce plant comprising mutagenizingthe part of plant of any one of claim 3 or
 4. 16. A method for producinga modified lettuce plant comprising mutagenizing the tissue culture ofclaim
 5. 17. A method of producing a lettuce seed comprising crossing amale parent lettuce plant with a female parent lettuce plant andharvesting the resultant lettuce seed, wherein said male parent lettuceplant or said female parent lettuce plant is the lettuce plant of claim2.
 18. An F1 lettuce seed produced by the method of claim
 17. 19. Alettuce plant produced by growing the seed of claim
 18. 20. A method forproducing a seed of a 45-IC3284 RZ-derived lettuce plant comprising (a)crossing a plant of lettuce variety 45-IC3284 RZ, a sample of seed ofwhich having been deposited under NCIMB Accession No. 44097, with asecond lettuce plant, and (b) whereby seed of a 45-IC3284 RZ-derivedlettuce plant forms.
 21. The method of claim 20 further comprising (c)crossing a plant grown from 45-IC3284 RZ-derived lettuce seed withitself or with a second lettuce plant to yield additional 45-IC3284RZ-derived lettuce seed, (d) growing the additional 45-IC3284 RZ-derivedlettuce seed of step (c) to yield additional 45-IC3284 RZ-derivedlettuce plants, and (e) repeating the crossing and growing of steps (c)and (d) for an additional 3-10 generations to generate further 45-IC3284RZ-derived lettuce plants, and (f) whereby seed of a 45-IC3284RZ-derived lettuce plant forms.
 22. A method of introducing at least onenew trait into a plant of lettuce variety 45-IC3284 RZ comprising: (a)crossing the plant as claimed in claim 2, with a second lettuce plantthat comprises at least one new trait to produce progeny seed, (b)harvesting and planting the progeny seed to produce at least one progenyplant of a subsequent generation, wherein the progeny plant comprisesthe at least one new trait, (c) crossing the progeny plant with a plantof lettuce variety 45-IC3284 RZ to produce backcross progeny seed, (d)harvesting and planting the backcross progeny seed to produce abackcross progeny plant, and (e) repeating steps (c) and (d) for atleast three additional generations to produce a lettuce plant of variety45-IC3284 RZ comprising at least one new trait and all of thephysiological and morphological characteristics of a plant of lettucevariety 45-IC3284 RZ, when grown in the same environmental conditions.23. The lettuce plant produced by the method of claim 22, wherein theplant comprises the at least one new trait and otherwise all of thephysiological and morphological characteristics of a lettuce plant oflettuce variety 45-IC3284 RZ.
 24. A method for producing lettuce leavesas a food product comprising sowing the seed of claim 1, growing theseed into a harvestable lettuce plant and harvesting the head or leavesof said plant, optionally processing and/or packaging the head or theleaves.
 25. A container comprising one or more of the lettuce plants ofclaim 2 for harvest of leaves.
 26. The lettuce plant of claim 2, whichis a plant grown from seed having been deposited under NCIMB AccessionNo. 44097.